We will determine the complete nucleotide sequences of the structural and regulatory regions of the tryptophan operons of E. coli and S. typhimurium. We will use this information to answer structural, functional, regulatory and evolutionary questions about these operons. We will produce intergeneric intragenic hybrid tryptophan synthetase alpha chains and assess the functional significance of the amino acid differences that exist in the alpha chains of E. coli and S. typhimurium. We will clone and study the structural gene and regulatory regions for tryptophan synthetase and kynureninase of Neurospora crassa. We will also clone and study the structural and regulatory regions for tryptophanase and tryptophanyl-tRNA synthetase (E. coli). We will determine whether the termination of transcription that is characteristic of operons with polar mutations (translational polarity) is due to unmasking of discrete transcription termination sequences within structural genes. We will construct an "ideal" E. coli trp operon and incorporate it onto a multicopy plasmid. We will study the pattern of in vivo degradation of the corresponding transcript. We will continue our study of the regulatory mechanism attenuation in an attempt to explain how translation of the initial region of the trp transcript controls transcription termination. The regulatory regions of trp operons of various Enterobacteria will be analyzed as well as the regulatory region of aroH of E. coli. The aporepressor that regulates transcription initiation on the trp operon of E. coli will be purified and its interaction with operator DNA studied.